Interactions between Natural Enemies of the Asian Citrus Psyllid in South Florida

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Interactions between Natural Enemies of the Asian Citrus Psyllid in South Florida
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english
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Navarrete, Bernardo
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Master's ( M.S.)
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University of Florida
Degree Disciplines:
Entomology and Nematology
Committee Chair:
Pena, Jorge E
Committee Members:
Mcauslane, Heather J

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Subjects / Keywords:
ants -- biological -- citri -- citrus -- control -- diaphorina -- intraguild -- longipes -- mutualism -- predation -- radiata -- tamarixia -- zelus
Entomology and Nematology -- Dissertations, Academic -- UF
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Abstract:
Two investigations were conducted to determine the interaction of natural enemies with Tamarixia radiata, an ectoparasitoid of Diaphorina citri (Hemiptera: Psyllidae) in Homestead, Florida. The first study focused on the effect of the predator Zelus longipes on the populations of D. citri and its parasitoid T. radiata under controlled conditions. Different densities of adults and 1st instar nymphs of Z. longipes were placed in experimental arenas with fixed numbers of D. citri nymphs, adults and T. radiata adults. Adults and nymphs of Z. longipes preyed on adults of D. citri and T. radiata. They also preyed on nymphs of D. citri but at a lower rate. Female Z. longipes adults preying on D. citri showed a Type III functional response. An experiment addressing prey preference showed that 1st instar nymphs of Z. longipes preferred to prey upon T. radiata adults over D. citri adults, while female Z. longipes preferred to prey upon Anastrepha suspensa adults over D. citri adults.The second study aimed to clarify the role of ants in D. citri biocontrol. This study was divided into two phases. During the first phase, we identified and observed the behavior of ants present in flushes of orange jasmine (Murraya paniculata) infested with D. citri during a 24-h period. The second phase focused on determining if ant presence affected parasitism of D. citri. In two experiments ants were excluded using Tanglefoot® as a physical ant barrier in both orange jasmine and Persian lime (Citrus latifolia). In an additional experiment the chemical bait Extinguish Plus® was used to control ants in Persian lime. The results showed at least four species of ants tending D. citri in south Florida. These species are: Brachymyrmex obscurior, B. patagonicus, Pheidole megacephala, and Solenopsis invicta. These ants are active day and night and were seen feeding on the honeydew excretions of D. citri nymphs. The results of the ant exclusion experiments showed that the percentage of parasitism by T. radiata was significantly higher in the flushes where ants were excluded using Tanglefoot® or Extinguish Plus®. The interaction between predators and parasitoids and the interference of ants in the performance of the parasitoid T. radiata might partially explain the lack of success of the biological control of D. citri in south Florida.
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In the series University of Florida Digital Collections.
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Includes vita.
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Thesis (M.S.)--University of Florida, 2012.
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Adviser: Pena, Jorge E.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-06-30
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by Bernardo Navarrete.

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1 INTERACTIONS BETWEEN NATURAL ENEMIES OF THE ASIAN CITRUS PSYLLID IN SOUTH FLORIDA By BERNARDO NAVARRETE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 Bernardo Navarrete

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3 To my mother and grandparents

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4 ACKNOWLEDGMENTS I would like to express my gratitude to Dr. Jorge Pea, my major Pr ofessor, for his continuous support, advice and assistance throughout my stay at the University of Florida. I would also like to thank Dr. Heather McAuslane, the other member of my Graduate Committee, for her helpful suggestions for the improvement of my r esearch. I thank Rita Duncan, Ana Vargas, Katia Santos and Jose Alegria for technical help during my research. Micah Gill of ARS/USDA, Miami, and George Schneider of DPI, Gainesville, for providing me with fruit flies for maintaining my assassin bug colony and Dr. Mark Deyrup of Archbold Biological Station for ants identification. Dr Daniel Carrillo for helpful review of my manuscript. My Master of Science studies in the University of Florida would have not been possible without the economic support of t he National Institute of Agriculture Research of Ecuador (INIAP). For this reason I thank the National Director, Dr. Julio Delgado, and the former Research Director, Dr. Jaime Tola, for giving me this opportunity. Finally my deepest words of gratitude go t o my family in Ecuador and USA for their constant support and love, especially to my Mom, Mariana, and to my uncles and aunts, Marlene, Victor, Ma. Luz, and Eloy.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 ABSTRACT ................................................................................................................... 10 CHAPTER 1 INTRODUCTION .................................................................................................... 12 Literature review ..................................................................................................... 12 The Asian Citrus Psyllid .......................................................................................... 12 Origin and distribution in the world ................................................................... 12 Hosts ................................................................................................................ 13 Biology .............................................................................................................. 13 Seasonality ....................................................................................................... 14 Greening disease ............................................................................................. 14 Biological control of D. citri ...................................................................................... 16 Worldwide overview ......................................................................................... 16 Biological control of D. citri in Florida ............................................................... 17 Tamarixia radiata .............................................................................................. 19 Zelus longipes .................................................................................................. 20 Intraguild predation ................................................................................................. 22 Ants and their role in biological control ................................................................... 24 Scope of the research ............................................................................................. 25 2 EFFECT OF Zelus longipes AGAINST Diaphorina citri AND ITS PARASITOID Tamarixia radiata UNDER CONTROLLED CONDITIONS ...................................... 27 Materials and Methods ............................................................................................ 29 Experiment 1. Effect of Several Densities of Z. longipes on Different Stages of D. citri Under Controlled Conditions .......................................................... 31 Experiment 2. Effect of Several Densities of Z. longipes on T. radiata Mortality. ........................................................................................................ 32 Experiment 3. Functional Response of Z. longipes as Predator of D. citri Adults ............................................................................................................ 33 Experiment 4. Prey Preference of Z. longipes ................................................. 34 Results .................................................................................................................... 35 Experiment 1. E ffect of Several Densities of Z. longipes on Different Stages of D. citri Under Controlled Conditions. ......................................................... 35 Experiment 2. Effect of Several Densities of Z. longipes on T. radiata Mortality. ........................................................................................................ 36

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6 Experiment 3. Functional Response of Z. longipes as predator of D. citri Adults, ........................................................................................................... 36 Experiment 4. Prey Preference of Z. longipes ................................................. 37 Discussion .............................................................................................................. 37 3 ANTS ASSOCIATED WITH Diaphorina citri AND THEIR ROLE IN ITS BIOLOGICAL CONTROL ........................................................................................ 50 Materials and Methods ............................................................................................ 52 Experiment 1. Identification and Behavior of Ants Related with D. citri in an Orange Jasmine Hedge. ............................................................................... 52 Experiment 2. Ant Exclusion Experiments in M. paniculata and C. latifolia ..... 53 Experiment 3. Effect of Ant Control on D. citri Parasitism. ............................... 54 Results .................................................................................................................... 54 Experiment 1. Identification and Behavior of A nts R elated with D. citri in an O range Jasmine H edge ................................................................................ 54 Experiment 2. Ant Exclusion Experiments in M. paniculata and C. latifolia ..... 55 Experiment 3. Effect of Ant Control on D. citri Parasitism. ............................... 56 Discussion .............................................................................................................. 57 4 CONCLUSIONS ..................................................................................................... 69 LIST OF REFERENCES ............................................................................................... 70 BIOGRA PH ICAL SKETCH ............................................................................................ 82

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7 LIST OF TABLES Table page 1 1 Various species of predators of Diaphorina citri. ................................................ 16

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8 LIST OF FIGURES Figure page 1 1 Number of Diaphorina citri adults dead found in treatments with different densities of Zelus longipes adults. ...................................................................... 41 1 2 Number of Diaphorina citri eggs in Murraya paniculata flushes in treatments with different densities of Zelus longipes ........................................................... 42 1 3 Number of D iaphorina ci tri adults dead and D citri eggs in M paniculata flushes in treatments with different densities of Z longipes 1st instar nymphs ... 43 1 4 Number of Diaphorina citri 5th instar nymphs dead found in treatments with different densities of Zelus longipes adults. ........................................................ 44 1 5 Number of Diaphorina citri 5th instar nymphs dead found in treatments with different densities of Zelus longipes 1st ins tar nymphs. ..................................... 45 1 6 Number of Tamarixia radiata adults dead found in treatments with different Zelus longipes adults densities. .......................................................................... 46 1 7 Number of Tamarixia radiata adults dead found in treatments with different Zelus longipes 1st instar nymphs densities. ....................................................... 47 1 8 Functional response of Zelus longipes adult females to increasi ng densities of Diaphorina citri adults. .................................................................................... 47 1 9 Preference of Zelus longipes 1st instar nymphs, males and females of Zelus longipes for Diaphorina citri and Tamarixia radiata adults. ................................. 48 1 10 Preference of males and females of Zelus longipes for Diaphorina citri and Anastrepha suspensa adults. ............................................................................. 49 2 1 Number of ants found i n Murraya paniculata flushes infested with Diaphorina citri during a 24h period. .................................................................................... 59 2 2 Behavior of ants registered in Diaphorina citri infested flushes. ......................... 60 2 3 Relationship between number of ants and number of nymphs of Diaphorina citri in Murraya paniculata ................................................................................... 61 2 4 Number of ants found in unprotected flushes ..................................................... 62 2 5 Proportion of ants species found in unprotected flushes in Citrus latifolia .......... 63 2 6 Number of Diaphorina citri nymphs present in flushes with and without ant exclusion treatment. ........................................................................................... 64

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9 2 7 Percentage parasitism by Tamarixia radiata in Diaphorina citri infested flushes with and without ant exclusion treatment ................................................ 65 2 8 Number of ants found in flushes of trees with and without the use of granular insecticidal ant bait in Citrus latifolia .................................................................. 66 2 9 Proportion of an ts species found in flushes of trees with and without the use of granular ant bait in Citrus latifolia .................................................................. 67 2 10 Number of Diaphorina citri nymphs present in flushes of trees with and without the use of granular ant bait in Citrus latifolia ........................................ 68 2 11 Percentage parasitism by Tamarixia radiata in Diaphorina citri infested flushes of trees with and without the use of granular ant bait in Citrus latifolia .. 68

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INTERACTIONS BETWE EN NATURAL ENEMIES OF THE ASIAN CITRUS PSYLLID IN SOUTH FLORIDA By Bernardo Navarrete December 2012 Chair: Jorge E. Pea Major: Entomology and Nematology Two investigations were conducted to determine the interaction of natural enemies with Tamarixia r adiata, an ectoparasitoid of Diaphorina citri (Hemiptera: Psyllidae) in Homestead, Florida. The first study focused on the effect of the predator Zelus longipes on the populations of D. citri and its parasitoid T. radiata under controlled conditions. Different densities of adults and 1st instar nymphs of Z. longipes were placed in experimental arenas with fixed numbers of D. citri nymphs, adults and T. radiata adults. Adults and nymphs of Z. longipes preyed on adults of D. citri and T. radiata. They also pr eyed on nymphs of D. citri but at a lower rate. Female Z. longipes adults preying on D. citri showed a Type III functional response. An experiment addressing prey preference showed that 1st instar nymphs of Z. longipes preferred to prey upon T. radiata adu lts over D. citri adults while female Z. longipes preferred to prey upon Anastrepha suspensa adults over D. citri adults.

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11 The second study aimed to clarify the role of ants in D. citri biocontrol. This study was divided into two phases. During the first phase, we identified and observed the behavior of ants present in flushes of orange jasmine ( Murraya paniculata) infested with D. citri during a 24h period. The second phase focused on determining if ant presence affected parasitism of D. citri. In two ex periments ant s were excluded using Tanglefoot as a physical ant barrier in both orange jasmine and Persian lime ( Citrus latifolia ) In an additional experiment the chemical bait Extinguish Plus was used to control ants in Persian lime. The results showed at least four species of ants tending D. citri in south Florida. These species are: Brachymyrmex obscurior B. patagonicus Pheidole megaceph ala, and Solenopsis invicta. These ants are active day and night and were seen feeding on the honeydew excretions of D. citri nymphs. The results of the ant exclusion experiments showed that the percentage of parasitism by T radiata was significantly higher in the flushes where ants were excluded using Tanglefoot or Extinguish Plus. The interaction between predators and parasitoids and the interference of ants in the performance of the parasitoid T. radiata might partially explain the lack of success of the biological control of D. citri in south Florida

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12 CHAPTER 1 INTRODUCTION Literature review The Asian Citrus Psyllid The Asian Citrus Psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is an invasive pest that arrived in Florida in 1998 (Hoy and Nguyen 1998) and since then has become a major threat for the Florida citrus industry, which represents 65% o f the total USA citrus production (USDA 2010). Nymphs and adults of this pest use their sucking mouthparts to extract the sap of citrus leaves and excrete honeydew that can serve as a substrate for sooty mold (Tsai 2008); while feeding, D. citri injects to xic saliva into its host causing leaf distortion (Michaud 2004). More importantly, D. citri transmits one of the most devastating diseases of fruit trees in the world, the greening or huanglongbing (Gottwald 2010). Origin and distribution in the world The Indian subcontinent seems to be the place of origin of D. citri (Hall 2008b). This species is distributed in several countries of continental Asia, including China, India, Pakistan, Iran, Afghanistan, Myanmar, Thailand, Malaysia, Nepal, Saudi Arabia, Yemen and the islands of Taiwan, Philippines, Indonesia, Sri Lanka, Ryukyu and Reunion, and Mauritius in Africa ( Waterhouse 1998, Halbert and Manjunath 2004, Talebi et al. 2011). The first records of the presence of D. citri on the American continent are f rom Brazil in 1942 (Hodkinson and White 1981) and Argentina in 1984 (Augier et al. 2006). According to EPPO (2010), D. citri is also in Uruguay and Paraguay and was reported in Colombia in 2011 (Ebratt Ravelo et al. 2011). Diaphorina citri dispersed quick ly through the Caribbean Islands since its detection in

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13 1998 (Guadeloupe, Abaco Island, Bahamas, West Cayman Island, Jamaica, Dominican Republic, Cuba, Puerto Rico and Venezuela) (Halbert and Nuez 2004). The first report of D. citri in USA was on the east coast of Florida (Broward to St. Lucie counties) in June of 1998 (Hoy and Nguyen 1998); and by September of 2000, it was found throughout 31 Florida counties (Halbert et al. 2001). Since July of 2010 this pest has been reported in Alabama, Arizona, California, Florida, Georgia, Guam, Hawaii, Louisiana, Mississippi, South Carolina, Texas, and the US Virgin Islands (Mead and Fasulo 2010). A recent genetic molecular study using mitochondrial cytochrome oxidase I (mtCO1) suggests the existence of two major haplotype groups of D. citri, one of southwestern Asian origin that includes the northern hemisphere populations (USA and Mexico) and an other of southeastern Asian origin (Boykin et al. 2012). Hosts Diaphorina citri is an oligophagous insect that only feeds o n plants belonging to the family Rutaceae. Most affect ed genera include Murraya, Citrus, Clausena, Triphasia, Fortunella, Poncirus, Merrillia, Vepris, Swinglea and Atalantia (Waterhouse 1998). Some citrus plants are better hosts than others. For instance, D. citri has a higher rate of development on grapefruit than on rough lemon, sour orange or orange jasmine, Murraya paniculata (L.) (Tsai and Liu 2000). However, Arredondo (2008) reported that lime and sweet orange were better hosts than grapefruit and mandarin. Orange jasmine is considered a key host plant for D. citri due to its continuous production of new flush (Tsai et al. 2002). Biology Diaphorina citri is a hemimetabolous insect with five nymphal stages. The nymphs are small (0.25 mm long in the 1st instar to 1.7 mm in the last instar) and their

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14 color is generally yellowish orange; the presence of big wing pads sets this species apart from other psyllids (Mead 1977). The adults are small (34 mm long) and can fly short distances when disturbed. Adult s are found feeding and resting on the leaves with their heads close the leaf surface and their bodies at a 45 angle from the leaf (Rogers et al. 2009). The immatures are found only on young tissue, whereas adults can feed on both new and old leaves (Tsai et al. 2002). Tsai and Liu (2000) studied the life cycle of D. citri on four host plants at 25C and determined that the duration of the egg stage could vary between 4.15 and 4.31 days, the first instar between 2.00 and 2.18 days, the second instar between 1.58 and 1.65 days, the third instar between 1.58 and 1.86 days, fourth instar between 2.32 and 2.46 days, and fifth instar between 4.65 and 5.50 days. The total life cycle (egg to adult) varied from 16.88 days on grapefruit to 17.32 days on sour orange. Seasonality Tsai et al. (2002) studied the seasonal abundance of D. citri in southern Florida. These authors reported population peaks in October, November, and December of 1998, and in May and August of 1999, and concluded that the population dynamics of psyllids is mainly influenced by the presence of new leaf flushes in the host (the flushing pattern is influenced by rainfall and low temperature) and that natural enemies did not contribute to regulation of the pest. Hall et al. (2008) determined that May, June, and July were the months with higher D. citri infestations in east central Florida citrus groves. Greening disease Diaphorina citri transmits the greening disease caused by the bacteri um Candidatus Liberibacter asiaticus which is considered on e of the most devastating

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15 diseases of citrus in the world (Halbert and Manjunath 2004). The first report of the greening transmission by D. citri is from India in 1967; at this time it was thought that greening was a viral disease (Capoor et al. 1967). Alt hough there is not absolute certainty about the origin of the greening disease, or Huanglonbing, the first records of greening like symptoms are from India (18th century) and China (19th century). Currently, the disease is found in Asia, Africa and Ameri ca (Da Graa 2008) Three phloem limited bacterial species are involved in the complex; Ca Liberibacter asiaticus and Ca L. americanus (transmitted by D. citri) and Ca L. africanus (transmitted by Trioza erytreae Del Guercio ) (Da Graa and Korsten 2004, Gasparoto et al. 2012) The first positive sample for citrus greening ( Ca L. asiaticus) in Florida was collected in Miami Dade County in 2005. By 2008, the disease has spread to all the citrus producing counties (Halbert et al. 2008). The symptoms of this disease start with leaf yellowing that may appear on a single shoot or branch; the leaves may have a mottled or blotchy appearance, and eventually the yellowing spreads throughout the tree causing dieback. The trees become unproductive in 23 years (Chung and Brlansky 2009). This disease induces very distinctive fruit symptoms, i.e., small size, asymmetry, curved axis, uneven color; this last characteristic is responsible for calling the disease greening (Bove 2006). Not all stages of D. citri can acqui re Ca L. asiaticus only fourth to fifth instar nymphs and adults can do so The pathogen can remain latent in the insect for a period of 2 to 25 days. Adults can transmit the bacteri um during their whole life (Xu et al. 1988). The rate of transmission is high when the psyllid acquires the bacterium as an immature stage (Inoue et al. 2009). The increasing amount of bacteria during the life

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16 stages of the psyllid could be a proof of the propagative nature of Ca L. asiaticus in D. citri (Hung et al. 2004). B iological control of D cit r i Worldwide overview Several predators, parasitoids and entomopathogenic fungi have been found affecting D. citri nymphs and adults in different places in the world. Most of the reported predators of D. citri are ladybugs (Coleoptera: Coccinellidae), lacewings larvae (Neuroptera: Chrysopidae) and Syrphid flies (Diptera: Syrphidae). Some members of these families reported in associat ion with D. citri are presented in the Table 11. Table 11. Various species of predators of Diap horina citri Order: Family Species Location Reference Coleoptera Coccinelidae Coccinella septempunctata L., C. rependa Thunberg, Cheilomenes sexmaculata F. Chilocorus nigrita F. Brumus suturalis F. India (Hussain and Nath 1927) Cheilomenes quadriplag iata Swartz, Coelophora biplagiata (Mulsant), Harmonia axyridis (Pallas), H. octomaculata (Fabricius), Propylea japonica (Thunberg) China (Yang et al. 2006) Cydonia propinqua (Mulsant), Cycloneda sanguinea L., Olla v nigrum (Mulsant) Mexico (Cortez Mondac a et al. 2010) Menochilus sexmaculatus (Fabricius), Scymmus levaillanti (Mulsant), and Exochomus nigripennis (Erichson) Iran (Rakhshani and Saeedifar 2012). Neuroptera Chrysopidae Chrysopa boninensis Okamoto and C. septempunctata Wesmael China (Yang et al. 2006) Chrysoperla comanche (Banks) Mexico Cortez Mondaca et al. 2010) C. carnea (Stephens) Iran (Rakhshani and Saeedifar 2012). Diptera Syrphidae Allobaccha sapphirina (Wiedeman) Iran (Rakhshani and Saeedifar 2012). Several species of Vespidae, Carabidae, Reduviidae and Histeridae have sporadically reported preying upon D. citri (Al Ghamdi 2000, Reyes Rosas et al. 2011) Some hunting spiders Zelotes sp. (Gnaphosidae) and Chirachantium sp. (Clubionidae) have been reported feeding on D. citri adult s in Iran (Rakhshani and Saeedifar 2012).

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17 The jumping spider Marpissa tigrina Tikader is also reported feeding on D. citri in India (Sanda 1991). Michaud (2002) found the species Hibana velox (Becker) (Anyphaenidae), Chiracantium inclusum (Clubionidae), He ntzia palmarum (Salticidae) and Oxyopes spp. (Oxyopidae) preying on D. citri in central Florida, USA. Pea et al. (2008) reported the orbweaver spider Eriophora ravilla Koch (Aranaeidae) preying on adults of D. citri on M. panic u l a ta and Citrus latifolia Tanaka in southern Florida. Diaphorina citri is a host of two parasitoids that have been used in classical biological control programs in Asia, Africa and America; these are the eulophid Tamarixia radiata (Waterston) and the encyrtid Diaphorencyrtus aligarhensis ( Shafee, Alam and Agarwal ) (Hall 2008a). Other reported parasitoids are less important and their role is not clear; many of them could be acting as hyperparasitoids (Tang 1990, Halbert and Manjunath 2004, Hall 2008a, Fallahzadeh and Japoshvili 2010). Several species of entomopathogenic fungi have been observed occurring naturally in D. citri colonies in places with high humidity, for example Cladosporium sp. nr. oxysporum Berk and M A Curtis and Capnodium citri Mont in Reunion (Aubert 1987), Hirsut ella citriformis Speare in Guadeloupe and Florida (Etienne et al. 2001, Meyer et al. 2008), Beauveria bassiana (Balsamo) Vuillemin in Cuba (Rivero and Grillo 2000) and Isaria fumosorosea Wize in Florida (Meyer 2009) Biological control of D. citri in Flori da Due to the importance of D. citri as a vector of the greening disease, a classical program of biological control was implemente d in Florida. Two specific parasitoids T. radiata and D. aligarhensis were collected, introduced and released in 1999 and 20 00 (Hoy et al. 2001). A survey made in 20062007 at locations in the central, southwest, and eastern coast showed that T. radiata was established in Florida but with lower

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18 percentages of parasitism than those observed in other countries. The mean percentag e of parasitism was less than 20% during spring and summer, increasing to 39% during fall, and reaching 56% in September (Qureshi et al. 2009). Chong et al. (2010) found an average of 18% parasitism in a survey on orange jasmine hedges in Miami Dade County Diaphorencyrtus aligarhensis does not appear to have been established in Florida (Michaud 2002, Rohrig et al. 2012). In a preliminary survey made in 2001, Michaud (2002) found that the main biological control was exercised by the coccinelids, O. v nigrum and H. axyridis Other predators seen feeding on D. citri are spiders (Anyphaenidae, Clubionidae, Oxyopidae and Salticidae), lacewings (Chrysopidae and Hemerobiidae), hoverflies (Syrphidae), and predatory bugs (Anthocoridae). The same author also conclud ed that the mortality of the psyllid due to T. radiata was not significant; a similar conclusion was reached by Tsai et al. (2002) who found less than 1% parasitism by T. radiata in southern Florida in 1999. In another experiment in central Florida, Michaud (2004) reported that protection of colonies of D. citri from major predators, i.e. H. axyridis and O. v nigrum spiders and lacewings, improved psyllid maturation success rate 120fold. Survival of the parasitoid T. radiata improved 40 old in the protect ed colonies. As proof of intraguild predation, he estimated that more than 95% of larvae and pupae of the parasitoid were consumed by predators. Similar results were found by Qureshi and Stansly (2009), who reported that in shoots protected from natural enemies, the net reproductive rate of D. citri was 5 to 27fold higher than in control shoots. The coccinellids, O. v nigrum, Curinus coeruleus Mulsant, H. axyridis and C.sanguinea, the cockroach Blatella asahinai Mizukubo, lacewings, Ceraeochrysa sp. and C hrysoperla sp., and spiders were the most common

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19 predators observed in this experiment; in consequence, the parasitism by T. radiata was generally low but was higher in the shoots protected by a sticky barrier suggesting the presence of intraguild predation. In south Florida, a survey for natural enemies showed that the assassin bug Zelus longipes ( L. ) was the most important predator of D. citri in orange jasmine hedges and lime orchards, followed by the syrphid, Allograpta obliqua (Say), the coccinellids, C. sanguinea and H. axyridis and the spiders, E. ravilla and Hentzia spp. The parasitoid T. radiata had a low percentage of parasitism suggesting an interaction with generalist predators. The ants Camponotus floridanus (Buckley) and Pheidole spp. were obs erved in the infested flushes but their role was not clear (Pea et al. 2008). The results of these experiments indicated that predators were the key mortality factor of D. citri in southern Florida. These authors reported that the parasitoid T. radiata is well established but their parasitism rate was lower than in other places like Puerto Rico (Pluke et al. 2008) and Reunion (Aubert et al. 1996). An additional survey conducted in M. paniculata hedges infested with D. citri in four locations in Miami Dade County showed an assemblage of general predators dominated by the coccinelids, H. axyridis Chilocorus stigma (Say) and C. sanguinea. In this research the percentage of parasitism by T. radiata fluctuated between 14 and 28% (Chong et al. 2010). Tamarixia radiata Tamarixia radiata (Hymenoptera: Eulophidae) is an idiobiont ectoparasitoid, that was first described from specimens caught in Lyallpur, Punjab, then India, now Pakistan (Waterston 1922). Even though D. citri is the main host of T. radiata this par asitoid has also been found attacking the psyllid Psylla hialina Mathur in India (Peter et al. 1990). The adult size range between 0.92 to 1.04 mm in length ; females are a little bit larger than males which have long and hairy antennae. The color of the he ad and thorax is

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20 shiny black, wings are hyaline but the color of the veins is pale yellow, and legs are almost entirely pale (Waterston 1922, Onagbola et al. 2008, Mann and Stelinski 2010, Qureshi and Stansly 2012). Females of this wasp lay their eggs beneath D. citri nymphs, between the thorax and the abdomen. Apparently, odor cues from D. citri nymphs are used by T. radiata to locate its host (Skelley and Hoy 2004). After hatching, the neonate larva starts feeding on the psyllids hemolymph until the deat h of the host (Mann et al. 2010, Mann and Stelinski 2010). When this occurs, the nymph turns dark brown in color having the appearance of a mummy (Hoy et al. 2006). The adult females also inflict mortality feeding on D. citri nymphs. According to Skelley and Hoy (2004), T. radiata can kill 57% of the psyllid nymphs by host feeding. An adult female can oviposit 166 to 330 eggs during its lifespan and each female has the potential to kill more than 500 psyllids nymphs adding the effect of host feeding and parasitism (Hoy et al. 2006). The fitness of T. radiata is highly influenced by temperature, with 25C being optimal for development and parasitism efficacy (Gomez Torres et al. 2012). At 25C the life cycle from egg to adult last 12 days (Skelley and Hoy 2004) Zelus longipes The assassin bug Z. longipes (Hemiptera: Reduviidae: Harpactorinae) is a predator with a broad range of prey Most reported prey are soft body insects like the psyllids, D. citri and Heteropsylla cubana Crawford ; the caterpillars, Ur esiphita reversalis (Guenee), Spodoptera frugiperda Smith, and Heliothis virescens F.; and the flies, Euxesta stigmatias Loew, E. eluta Loew and E. annonae F. (Valenciaga et al. 1999, Carrel 2001, Alvarez Hernandez et al. 2002, Pea et al. 2008, Kalsi 2011). Zelus longipes forages in a diversity of plants in which its prey are located; it is common in row crops like corn, but also in shrubs and weeds. According to Berenger and Pluot Sigwalt

PAGE 21

21 (1997), members of the Harpactorinae like to forage on plants that could provide them with gluey substances that they use to set up sticky traps. This predator has been seen as the most frequent natural enemy of D. citri in Homestead, Fl. (Pea et al. 2008). In preliminary observations made by the author, the number of adults of Z. longipes recorded during sets of 30 min observations in a 3 0 m long M. paniculata hedge fluctuated between 29 and 75 individuals/set on different evaluation dates. Linnaeus described Z. longipes as Cimex longipes in 1767 using specimens from the Island of Saint Thomas in the West Indies. During 1872 Stl changed its name to Z. longipes (Hart 1986, Maldonado Capriles 1990). The genus Zelus is probably native to Central and South America (Hart 1987); with the exception of Chile, Z longipes is w idely distributed from the southern United States to Argentina. Zelus longipes has the following biological stages: egg, five instar nymphal stage s, and adult (Melo et al. 2005). Amaral Filho and Fagundes (1996) reported that eggs develop within ca. 16.4 days, nymphs last 48.4 days and adults have a longevity of 40 days for females and 24.5 days for males. According to Kalsi and Seal (2011), adults of Z. longipes can be separated from other congeners based on characteristics of the pronotum (humeral angles unarmed and rounded), color of the dorsal surface (brownish red to brownish black), and male genitalia (parameres cylindrical and long). Zelus longipes and all the members of the Zelus genus have two strategies for catching their prey: stalking slow moving insects and using a sticky trap strategy for flying insects. The sticky trap strategy consists of raising their forelegs which are covered with a sticky substance, until the prey gets stuck in it (Edwards 1966). According to Wolf and Reid (2001), the t ibia of the forelegs of Z. longipes had a special

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22 system of hairs and peg like setae that work together in order to stabilize the film of a sticky substance present on the legs. This predator uses an extraoral digestion system to acquire its food (Cohen 1998, Cogni 2002). Intraguild predation Usually herbivores are exposed to the action of a guild of natural enemies. There are three possible outcomes when more than one natural enemy is preying in the same herbivore. First natural enemies can act synergis tically, increasing the rate of mortality. Second, they might not interact; in this case the total mortality infringed on the prey is the consequence of the sum of each individual mortality. Third, they can inter fere in such a way that the total mortality is less than the sum of each individual mortality (Ferguson and Stiling 1996). This last scenario is a complex relationship between carnivorous insects known as intraguild predation, a phenomenon that occurs when two predators that share the same prey, als o have a trophic relationship between them (Speight et al. 2007). Usually the intraguild predator is an omnivore that is able to eat more than one kind of prey in the same trophic level and the intraguild prey can be a primary predator or a parasitoid. In most of the cases, intraguild predation leads to a disruption of the biological control of the herbivore (Rosenheim et al. 1993). One example is the study of Ferguson and Stiling (1996), which compared different combinations of predators and parasitoids of aphids in field cages on marsh elder. They found that the presence of coccinellids interfered with the performance of braconid parasitoids and concluded that, in this case, the use of the two organisms would have a negative effect on the regulation of the pest population. Similar results were found by Zang and Liu (2007) with Delphastus catalinae Horn, a predator that interferes with the activity of Encarsia sophia (Girault and Dodd), a parasitoid of Bemis i a tabaci

PAGE 23

23 Gennadius. Moreover Rosenheim et al. (199 3) demonstrated that intraguild predators, i.e., the generalist hemipterans ( Geocoris sp., Nabis sp. and Zelus sp.) prey upon the intermediate predator Chrysoperla carnea (Stephens) and diminished its population to such an extent that its efficacy in the biological control of Aphis gossypii Glover in cotton was reduced significantly. On the other hand, the existence of intraguild predation is not always a guarantee of failure of biological control. For example, Colfer and Rosenheim (2001) demonstrated that Hippodamia convergens GurinMneville preying on parasitized mummies of A. gosypii did not have consequences on the success of the regulation of this pest in cotton. Another valid consideration is the possibility of coexistence of predators and parasitoids without intraguild predation For instance, Serangium japonicum Chapin, a coccinelid predator of B. tabaci is able to discriminate between parasitized and nonparasitized nymphs, preferring to eat the latter nymphs (Fazal and Xiang 2004). Lucas (2005) r eviewed the information available about intraguild predation among aphidophagous predators. He concluded that temporal and spatial distribution of aphids favor the existence of this kind of interaction. He identified three trophic levels and four major players in the intraguild predation scheme in aphidophagous predators. The protagonists are: intraguild predator, intraguild prey, extraguild prey (herbivore) and plant, and possibly ants (Hymenoptera: Formicidae) modulating the interaction between other predators. Being that aphids and psyllids are similar in their temporal and spatial distribution and having a similar guild of predators, it is possible that the complex interactions occurring with the natural enemies of aphids, also occurs with psyllids.

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24 Ant s and their role in biological control Ants are eusocial insects with a very wide range of feeding preferences; some of them are carnivorous, others herbivorous or fungivorous and some feed on sap, nectar, honeydew or related substances (Triplehorn and Johnson 2005). The carnivorous ants are important biological control agents. For instance, the first known report of the utilization by man of an insect as a predator was the use of the ant Oecophylla smaragdina Karavaev and Karawajew for the control of cater pillars and pentatomids in citrus in China during 420 A.D.(Huang and Yang 1987). However, Vandermeer et al. (2002) caution that the use of ants as biocontrol agents is restricted due to their habit to tend honeydew producing hemipteran pests, and also for their omnivorous feeding, that is a threat for other natural enemies. Eubanks (2001) made a correlation between the presence of Solenopsis invicta Buren and the population of pest and natural enemies in cotton and soybean. His results suggest ed that S. inv icta plays an important role in the biological control of most of the pests in these two crops, but it also interferes with the population of many natural enemies, such as spiders. A complementary research study made in cotton found strong evidence of the negative effect of fire ants on lady beetles ( Coccinella septempunctata L. and H. convergens ) and green lacewing larvae ( C. carnea) (Eubanks et al. 2002). Ants can also disrupt the biological control of the parasitoid Lipolexis scutellaris Mackauer against the brown citrus aphid Toxoptera citricida (Kirkaldy). Hill and Hoy (2003) determined that S. invicta reduces the populations of L. scutellaris because ants selectively remove and prey on parasitized aphids. Interestingly, according to Novak (1994), ants can also have a positive effect on the population of parasitoids by attacking hyperparasitoids. He arrived at this conclusion based on an experiment with ants tending psyllids; the ant tended psyllid Cacopsylla

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25 crateagui (Schrank) had higher levels of para sitism than other nonattended psyllids like C. peregrina Forster and C. melanoneura Forster. A similar situation was observed by Kaneko (2002) with the ant Lasius niger (L.) tending A. gossypii parasitized by Lysiphlebus japonicus Ashmead. In some ant hem ipteran pest systems the level of interference with the biological control is so high that methods of ant control are needed in order to reduce the population of the pest. That is the case of the ants tending the vine mealybug Planococcus ficus (Signoret) in California and South Africa. Klotz et al. (2003) found that direct sprays of Lorsban at the base of the vines were effective in reducing populations of Formica perpilosa (Wheeler) for nine weeks in California. In South Africa chlorpyrifos impregnated bands used against the ants Linepithema humile (Mayr) and Anoplolepis custodiens (Smith), result ed in a three month field efficacy (Addison 2002). Some species of ants live in association with D. citri because they feed on the honeydew excreted by the nymphs The following are reported to be associated with D. citri in Florida: Brachymyrmex obscurior Forel, Camponotus floridanus (Bucley), Crematogaster ashmeadi (Mayr), Dorymyrmex bureni (Trager), Monomorium floricola Jerdon, Paratrechina bourbonica (Forel), P seudomyrmex gracilis (Fabricius), S. invicta and Pheidole spp. Some of them have been observed carrying D. citri nymphs but their exact role in the biocontrol of D. citri is not clear and warrants further research (Michaud 2002, 2004; Pea et al. 2008). S cope of the research A common pattern in the results of biological control research of D. citri in south Florid a is the presence of intaguild predation generalist predators and the lack of clarity of ant s function in the system. This thesis aimed to gener ate more information on this

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26 subject. T he second chapter present s the results of a series of experiments carried out under controlled conditions to evaluate the effect of different densities of Z. longipes against D. citri and its parasitoid T. radiata. Th e third chapter deals with the role of ants on the biological control of D. citri. I tried to answer this question through several observations and ant exclusion experiments in M. paniculata and C. latifolia

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27 CHAPTER 2 EFFECT OF Zelus longipes AGAINST D iaphorina citri AND ITS PARASITOID Tamarixia radiata UNDER CONTROLLED CONDITIONS The assassin bug Zelus longipes ( L .) (Hemiptera: Reduviidae: Harpactorinae), is a generalist predator of soft bodied insects (Kalsi and Seal 2011). Examples of its reported are the psyllids Diaphorina citri Kuwayama (Pea et al. 2008), a herbivore of species in the family Rutaceae, Heteropsylla cubana Cra w ford w hich feeds on Leucaena leucocephala (Lam) (Valenciaga et al. 1999), and Uresiphita reversalis (Guenee) which feeds on the sky blue lupine Lupinus cumulicola Nutt (Carrel 2001) in the Fabaceae. Additional hosts include larvae of the lepidopterans, Spodoptera frugiperda Smith (Cogni et al. 2002, Hoballah et al. 2004) and Heliothis virescens F. (Alvarez Hernandez et al. 2002), and the dipterans, Euxesta stigmat i a s Lo e w, E. eluta Loew, and E. annonae F. (Kalsi 2011) that inhabit row crop agroecosytems such as corn. Zelus longipes also has been found preying on insects on broad leaf weeds such as Baccharis halimifolia L. (Palm er and Bennett 1989), Chenopodium album L., and Amaranthum tricolor L. (Rodriguez et al. 2005), on the grasses Pennisetum purpureum cv. Mott and Tripsacum laxum and in Vicia faba L. (Nuessly et al. 2004). This predator is found on many unrelated plant species. According to Berenger and Pluot Sigwalt (1997), members of the Harpactorinae are known to inhabit plants that provide them with viscous substances that are used by the bugs to set up their sticky traps. The genus Zelus is probably native to Central and South America (Hart 1987); Zelus longipes is widely distributed from the southern United States to Argentina, with the exception of Chile. The first description of the species was made by Linnaeus using specimens from the Island of Saint Thomas in th e West Indies (Hart 1986).

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28 All the species of the genus Zelus including Z. longipes have a common preying behavior consisting of two strategies: 1) stalk slow moving insects, and 2) catch flying insects, using a sticky trap strategy that consists of r aising its forelegs that are covered with a sticky substance, until the prey get stuck in it. After that the predator inserts its stylets, extending the rostrum, and the prey is quickly paralyzed (Edwards 1966). According to Wolf and Reid (2001), the tibia of the forelegs of Z. longipes ha ve special system of hairs and peg like setae that work together in order to stabilize the film of sticky substance present on the legs. This predator uses an extraoral digestion system to acquire its food, injecting high ly potent digestive enzymes which cause liquefaction of internal structures of the prey, that are sucked back into predators gut (Cohen 1998, Cogni et al. 2002) In an experiment using different sizes of S. frugiperda larvae as a food supply, this predator showed preference for small prey (Cogni et al. 2002). In another study Kalsi (2011) showed that Z. longipes displayed a Type II functional response to adults of three species of picture winged flies that feed on corn ( Euxestia stigmatias, E. eluta and E. annonae). Zelus longipes has been reported preying upon D. citri on M. paniculata in central east Florida (Hall et al. 2008) and in s outh Florida (Pea et al. 2008). In central east Florida Z. longipes was seen occasionally feeding upon D. citri but in So uth Florida it was a very common predator. This was noted by the author in preliminary observations, in which the number of adults of Z. longipes seen in 30 min of observation in a 3 0 m long M. paniculata hedge, fluctuated between 29 and 75 individuals on different evaluation dates and it was the most frequent predator present in the study

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29 site. In this survey, Z. longipes was seen feeding on D. citri but also on coccinelids, tachinid flies, and small wasps. Pea et al. (2008) anecdotally found Z. longipes eating adults of Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) which is a highly specific parasitoid, that has been released in Florida for the biological control of D. citri (Hoy and Nguyen 1998). Observations made by the author during the rearing of Z. longipes confirm that this reduviid feeds upon the Caribbean fruit fly Anastrepha suspensa (Loew), a tephritid that can affect citrus in Florida (Swanson and Baranowsky 1972). These preliminary data demonstrated the need for more information on the predation efficacy on this insect species and on its potential effect upon other biological control agents present in the system. Therefore, the research presented here has as objectives: 1) evaluate the effect of different densities of Z. longipes on D. citri under controlled conditions, 2) evaluate the effect of different densities of Z. longipes on T. radiata adults under controlled conditions, 3) determine the functional response of Z. longipes when using D. citri adults as prey, 4) determine the p rey preference of Z. longipes using D. citri, T. radiata and A. suspensa (Diptera: Tephritidae) as prey. Materials and Methods Stock Colonies : Zelus longipes adults and 1st instar nymphs used in the experiments described below were reared at the Tropical R esearch and Education Center Insectary (TREC) of the University of Florida, Homestead, FL. Adults of both sexes collected on a M. paniculata hedge at TREC were placed in a Plexiglass cage (303030 cm) with 200 cm2 of corrugated cardboard that served as an oviposition substrate. Honey was supplied on two pieces of paper (55 cm) fixed to the cage walls. Water was provided in a clear plastic container, with a cotton roll inserted through the container lid hole to allow predators access to water.

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30 Fifteen days after oviposition, 1st instar individuals emerged. Nymphs were daily fed with adults of Drosophila spp. (13 flies per individual) until the 4th instar was reached. Fourth instar specimens were then fed daily with adults of A. suspensa (1 3 flies per Z. l ongipes ). Adults of Z. longipes were obtained 45 to 60 days after the 1st instar emergence. Drosophila spp. was reared using ripe banana fruits kept in 1L plastic container and adults of A. suspensa were supplied by the Division of Plant Industry from th e Biocontrol Rearing Facility at the Florida Department of Agriculture and Consumer Services, in Gainesville, FL. Diaphorina citri adults and nymphs were obtained from a 3 0 m long M. paniculata hedge located at TREC. Adults were collected using an aspirat or and nymphs were collected by cutting infested flushes. Vials with adults and plastic bags with the infested flushes were placed inside a cooler and brought to the laboratory. The insects were immediately used in the experiments. Parasitoids : M urraya paniculata flushes (10 cm long) infested with 4th and 5th instar nymphs of D. citri were placed in plastic containers with 74 m L of water and then placed inside a Plexiglas cage (303030 cm) with adults of T. radiata Containers were maintained in an insect ary at 25 2C, 12:12 (L: D) and 65 2% RH. Honey and water were provided in the same way as it was done for Z. longipes Once we were certain that D. citri nymphs were parasitized, flushes containing the parasitized nymphs were transferred to a new cag e. Parasitoids began to emerge from the parasitized flushes after 12 to 15 days. This procedure was repeated every 10 days.

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31 Plant material : The flushes used in the experiments described below were obtained from M. paniculata plants that were grown in 3.8L plastic pots with a media of processed pine bark, 60% + Canadian sphagnum peat, and vermiculit e. The plants were put in 91.4 61 cm rearing cages (mesh size ~48 48) inside a greenhouse to avoid insect infestations. The experimental arena for the following trials was a mason jar (Ball 32 Oz wide mouth) sealed after placing the insects inside with a fine screen (4 holes per 1 mm2) mesh. A piece of wet cotton was placed inside with the objective of providing moisture. All the treatments were repeated f ive times. Food was withheld for 24 h for all the predators before being used in the experiment Experiment 1. Effect of S everal D ensities of Z. longipes on D ifferent S tages of D. citri U nder C ontrolled C onditions Adults of Z. longipes vs. adults of D. cit ri : Four Z. longipes densities (1, 2, 4, and 8 1d old adults of Z. longipes per arena) were exposed to 20 adults of D. citri inside experimental arenas provided with two 10cm long M. paniculata flushes. The experiment was cond ucted with Z. longipes females and males separately. Experimental arenas with D. citri but no predators were used as a control treatment. Twenty four hours later, the flushes were inspected under the stereoscope and the number of D. citri eggs per flush, t he number of dead D. citri and Z. longipes adults counted. 1st instar nymphs of Z. longipes vs. adults of D. citri : Five densities of 1st instar nymphs of Z. longipes (1, 2, 4, 8 and 12 1 d old 1st instar nymphs of Z. longipes per arena) were exposed to 2 0 adults of D. citri inside experimental arenas provided with two 10cm long M. paniculata flushes Control

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32 treatments wer e included as described above. The response variables were the number of D. citri dead, the number of Z. lo ngipes nymphs dead, and the number of D. citri eggs per M. paniculata flush. Adults of Z. longipes vs. nymphs of D. citri : This experiment had the same methodology and treatments as the first experiment, but in this case the prey were 20 5th instar D. cit ri nymphs on a 10cm M. paniculata flush. A sub treatment was the sex of the predators, as both Z. longipes males and female adults were tested. Twenty four h after exposure to the predators, the numbers of dead or alive D. citri and Z. longipes nymphs wer e evaluated under a microscope. 1st instar Z. longipes nymphs vs. D. citri nymphs: This experiment followed the same methodology of 1.3 but the treatment s were 0, 1, 2, 4, 8, and 12 1d old 1st instar nymphs of Z. longipes per arena. Experiment 2. Effect o f S everal D ensities of Z. longipes on T. radiata M ortality Z elus longipes adults vs. T. radiata adults: A 1 d old Z. longipes female and 20 1d old T. radiata adults were placed in the experimental arena. Twenty four hours later, the number of live and dead adults of both predator and prey was counted. The same procedure was repeated with 2, 4, and 8 1d old female adults of Z. longipes The untreated control treatment followed a similar methodology, but Z. longipes was excluded. The response variables were the number of T. radiata adults dead and number of surviving Z. longipes adults. The same experiment was repeated using 1d old male adults of Z. longipes Z elus longipes nymphs vs. T. radiata adults : This experiment followed the same methodology of 2.1 but in this case, the treatments were 0, 1, 2, 4, 8, and 12 1d -

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33 old 1st instar nymphs of Z. longipes per arena. The response variables were the number of dead T. radiata adults, and dead Z. longipes nymphs. Statistical Analysis: The response variables were analyzed using a oneway analysis of variance using the function PROC GLM (SAS 200 1 ). Tukeys test was used to determine significant differences between means ( P <0.05). T he data of the variable number of eggs of D. citri in experiments 1.1 and 1.2 were not normally distributed. Graphical analysis of the residuals using PROC GPLOT (SAS 200 1 ), indicated that a Experiment 3. Functional Response of Z. longipes as Predator of D. citri Adults In this experiment six D. citri adul t densities (4, 8, 12, 16, 20, and 24 per arena) were offered to a single 1d old Z. longipes female adult Both prey and predator were placed in the experimental arena with one flush of M. paniculata. Twenty four hours later, the numbers of live and dead adults of D. citri were counted. Each prey density was replicated six times. An untreated control for each density consisted of the prey without the predator. The type of functional response was determined using the polynomial logistic regression model (SA S Institute 200 1 ). Where Ne is the number of prey eaten, No the initial number of prey, and P0 P1 P2 and P3 are parameters estimated in the model. The handling time and attack constant were estimated using the NLIN procedure (Juliano 2001) with the Hassell equation as a model (Hassell 1978): = { 1 exp [( + ) ( ) /( 1 + ) ]} 0 = ( 0 + 1 0 + 2 0 2 + 3 0 3 ) 1 + ( 0 + 1 0 + 2 0 2 + 3 0 3 )

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34 In this equation, a is the attack constant, b ,c and d are constant s, Th is handling time and T is total time available for Z. longipes to search and attack the D. citri adults. Experiment 4. Prey Preference of Z longipes Prey preferences of 1st instar Z. longipes nymphs, female and male adults were determined by choice tests. The prey species were T. radiata, D. citri and A. suspensa adults. First, one flush of M. paniculata containing 15 adults of D. citri. plus 15 adults of T. radiata w ere introduced into the experimental arena followed by the introduction of either a 1st instar Z. longipes or a n adult male or female Z. longipes The arena was sealed thereafter. Twenty four hours later, the numbers of live and dead adults of D. citri and T. radiata were counted. Each experimental unit was replicated 15 times. The index proposed by Manly et al. (1972) was used to quantify the prey preference. = ln ( ) ln ( ) = 1 ( 1 + ) Where N and N are the numbers of each provided prey and Ne and Ne are the numbers of each prey killed. The preference index assigns values from 0 to 1, where 0.5 represents no preference. Mean values were considered significant when 9 5% confidence intervals based on the t distribution did not overlap with = 0.5. The same methodology was used for a separate experiment in which adult Z. longipes (male and female) had to choose between 15 adults of A. suspensa and 15 adults of D. citri.

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35 Results Experiment 1. Effect of Several Densities of Z. longipes on Different Stages of D. citri U nder Controlled Conditions Densities of two to eight Z. longipes adult females per arena ( F =117.58; df = 4, 20; P <0.001), and males ( F =28.77; df = 4, 20; P <0.001) caused higher mortality of D. citri adults than did a single male or female Z. longipes (Fig. 1 1). Regardless of the predator densities, the number of D. citri eggs deposited by surviving females was drastically reduced ( F =6.76; df = 4,20 P =0.0 013) when Z. longipes females was present compared with the number of D. citri eggs when it was excluded. However, Z. longipes males only caused a significant reduction ( F =6.06; df = 4,20 P =0.0023) of D. citri eggs when densities of four to eight predators were present in the arena as compared with the untreated control (Fig. 12). Zelus longipes nymphs were only effective as predators of D. citri adults at predator densities of eight and 12 per arena ( F =25.72; df = 5,24 P <0.001), it was observed that Z longipes nymphs usually prey on groups of two or more predators per single prey. No significant differences ( F =2.55; df = 5,24 P =0.0548) were observed on the number of D. citri eggs deposited in the M. paniculata flushes when two to eight Z. longipes n ymphs per arena were present (Fig.13). The effect of Z. longipes against nymphs of D. citri was not as remarkable as their effect on adults of D. citri. Densities of four to eight Z. longipes females per arena consumed more D. citri nymphs than did a sin gle Z. longipes female. The densities of two, four and eight Z. longipes females per arena resulted in more D. citri dead nymphs compared with the untreated control ( F =30.44; df = 4,20 P <0.001). Four to eight Z. longipes males per arena caused the highes t number of dead D. citri nymphs compared

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36 with the results obtained at lower predator densities or in the untreated control ( F =9.14; df = 4,20 P = 0.0005) (Fig.1 4). Only the treatment with eight Z. longipes nymphs could reduce significantly the number of D. citri nymphs ( F =3.14; df = 5,24 P =0.0256) (Fig.15). The mortality of Z. longipes was zero in all the experiments. Experiment 2. Effect of S everal D ensities of Z. longipes on T. radiata M ortality Adults and nymphs of Z. longipes fed upon T. radiata a dults. All male ( F =93.98; df = 4,20 P <0.001) and female ( F =318.71; df = 4,20 P <0.001) (Fig.16) adult predator densities tested resulted in mortality of nearly all the parasitoids offered. Mortality of the adult parasitoids increased with higher densit ies of Z. longipes nymphs and densities as low as two nymphs per arena caused significantly more T. radiata mortality than occurred when the predator was excluded ( F =28.77; df = 4,20 P =<0.001) (Fig.17). The mortality of Z. longipes was zero in both experiments. Experiment 3. Functional Response of Z. longipes as predator of D. citri Adults The binomial logistic regression analysis used to determine the shape of the functional response resulted in the following coefficient estimates: intercept ( a ) = 1.17 42 1.57 (SE), 2 = 0.56, P = 0.4545; linear ( b ) = 0.8682 0.4106(SE), 2 = 4.47, P = 0.003; quadratic ( c) = 0.0621 0.0304(SE), 2 = 4.17, P = 0.041 and cubic (d) = 0.00125 0.00067(SE), 2 = 3.45, P = 0.0631. The linear coefficient ( b ) was > 0 indicatin g that adult female Z. longipes showed a Type III functional response. The model produced the following values for the parameters of the Hassel equation: The handling time ( Th ) was 1.15 0.08 h and the attack rate constant ( a ) was 0.017 0.004 (Fig. 1 8)

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37 Experiment 4. Prey Preference of Z. longipes The first experiment indicated that Z. longipes 1st instar nymphs have a significant preference for T. radiata adults (95% confidence intervals did not include =0.5) over D. citri adults. Neither adult sex of Z. longipes showed any preference for the prey offered (Fig. 19). In the second experiment Z. longipes females showed a significant preference for A. suspensa adults (95% confidence intervals did not inclu de = 0.5) when compared with D. citri adults. The males did not show any preferences for the prey offered (Fig. 1 10). Discussion The results found in this research suggest that Z. longipes should be able to reduce the population of D. citri in orange jasm ine. Adults and nymphs consumed the adults of D. citri and reduced psyllid egg deposition in the M. paniculata flushes, reducing the possibility for build up a new pest generation. This confirms the observations by Pea et al. (2008) and Hall et al. (2008) about the association between Z. longipes and D. citri. Females were slightly more efficient predators of D. citri adults than males, which can be explained by the fact that Z. longipes females are larger than males and perhaps need more prey than the males (Kalsi 2011). Due to their small size, 1st instar nymphs of Z. longipes were able to reduce psyllid population to a lesser extent than the adults; only the highest densities of eight and 12 nymphs were effective. Another fact that may explain the smal ler number of D. citri eaten by Z. longipes nymphs is that groups of two or three Z. longipes nymphs often attacked a single prey. As a result, an individual nymph consumed fewer psyllids compared to the number

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38 consumed by Z. longipes adults. A similar beh avior was reported with Zelus exsanguis Stl attacking Ephestia kuehniella Zeller and Drosophila spp. (Edwards 1966). Immatures and adults of Z. longipes fed upon 5th instar nymphs of D. citri but the number of individuals consumed was very low in comparis on with the number of D. citri adults consumed. Apparently Z. longipes prefers to eat mobile prey instead of sessile prey. These results are in agreement with the results from a behavioral study with Z. renardii Kolenati (Cisneros and Rosenheim 1998); thes e researchers found that visual stimuli seem to be the most important cue needed for the predator to trigger an attack. Similar results were also obtained by Haridass et al. (1988) with the reduviid Rhinocoris marginatus F. In contrast, other Harpactorinae like Scipinia r e pax Stl and Nagusta sp., which are specialist predators of spiders, seem to rely on more sensory cues for orienting to prey (Jackson et al. 2010). In the generalist, Sinea diadema F., antennal olfaction appears to be more important than v ision for prey location (Freund and Olmstead 2000). Tamarixia radiata adults were suitable prey for adults and nymphs of Z. longipes If this behavior is confirmed in the field, it would imply an intraguild predation relationship that can disrupt the biol o gical control of the psyllid. And could also explain low levels of parasitism observed by Pea et al. (2008) in the same study site. Kalsi and Seal (2011) mentioned that Z. longipes can be a predator of other natural enemies like Orius spp. Zelus renardii has been reported as an important predator of parasitoids of the genus Aphytis contributing to the disruption of the biological control of the California red scale Aonidiella aurantii (Maskell) (Heimpel et a l. 1997). The same predator was

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39 also responsible for an increase of aphid populations due to the co nsumption of lacewings in intraguild predation experiments (Rosenheim et al. 1993). I did not observe cannibalistic behavior among Z. longipes individuals in the present experiments. This could be due to t he short duration of the trial (24 h). According to personal observations in the Z. longipes colony, cannibalism starts after 3 d of starvation. Similar behavior was observed by Evans (1976) with the pirate bug Anthocoris confuses (Reuter). Zelus longipes female adults had a type III functional response to adults of D. citri. In this type of functional response the proportion of prey killed by the predators initially increase and then decrease as the number of prey increases, creating a sigmoidal curve when plotting prey density against prey attacked (Juliano 2001); Type III functional response is typical for generalist predators that can switch from one prey to another (Hassell 1978, Akre and Johnson 1979, van Baalen et al. 2001). Kalsi (2011) found that both genders of Z. longipes adults had a type II functional response when feed upon flies of the genus Euxesta. The different types of functional responses for the same predator species may be explained by the use of different prey with different behavior and size. It is generally accepted that reduviids do not have food preferences because most of them are generalist predators which feed on the prey that they randomly encounter while they are foraging (Louis 1974). The experiments dealing with prey preferen ces of Z. longipes adults confirm this assumption because we did not find a preference for the prey offered ( D. citri and T. radiata); however Z. longipes 1st instar nymphs had a preference for T. radiata adults over D. citri adults. This result can be

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40 exp lained by the smaller size of the parasitoid in comparison with the psyllid. For instance, Cogni et al. (200 2 ) determined in choice experiments that Z. longipes prefers smaller prey. In the last experiment, the adult females of Z. longipes showed a prefere nce for A. suspensa adults over D. citri adults; in this case the females chose a bigger prey, which can be explained by the different nutritional needs of the females regarding their reproductive role or by their bigger size in comparison with males. The results of this research indicated that Z. longipes adults may prey upon adults of D. citri outdoors in south Florida. Considering the high populations of this reduviid seen in south Florida and the functional response showed toward the psyllid, it is reas onable to think that this predator has a great potential for controlling D. citri. However it was also demonstrated in this research that the parasitoid T. radiata, is a suitable prey for Z. longipes The prey preference estudies showed that 1st instar ny mphs had a preference for the smaller parasitoid over the larger D. citri. For this reason the real effect of th is predator on psyllid population should be tested under field conditions before considering it as a candidate in a biological control program.

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41 Figure 11. Number of Diaphorina citri adults dead found in treatments with different densities of Zelus longipes adults. Females (A) and males (B). Treatments with different letters are significantly different according to the Tukeys Test ( P <0. 05). Error bars represent the standard error of the mean.

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42 Figure 12 Number of Diaphorina citri eggs found in Murraya paniculata flushes in treatments with different densities of Zelus longipes adults. Females (A) and males (B). O riginal data trans Treatments with different letters are significantly different according to the Tukeys Test ( P <0.05). Error bars represent the standard error of the mean.

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43 Figure 13. Number of Diaphorina citri adults dead and D citri eggs in M panic ulata flushes in treatments with different densities of Zelus longipes 1st instar nymphs Adults (A) and eggs (o ) (B). Treatments with different letters are significantly different according to the Tukeys Test ( P <0.05). Error bars represent the standard error of the mean.

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44 Figure 14. Number of Diaphorina citri 5th instar nymphs dead found in treatments with different densities of Zelus longipes adults. Females (A) and males (B). Treatments with different letters are signi ficantly different according to the Tukeys Test ( P <0.05). Error bars represent the standard error of the mean.

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45 Figure 15. Number of Diaphorina citri 5th instar nymphs dead found in treatments with different densities of Zelus longipes 1st instar nym phs Treatments with different letters are significantly different according to the Tukeys Test ( P <0.05). Error bars represent the standard error of the mean.

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46 Figure 16. Number of Tamarixia radiata adults dead found in treatments with different Zelus longipes adults densities. Females (A) and males (B). Treatments with different letters are significantly different according to the Tukeys Test ( P <0.05). Error bars represent the standard error of the mean.

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47 Figure 17. Number of Tamarixia radiata adults dead found in treatments with different Zelus longipes 1st instar nymphs densities. Treatments with different letters are significantly different according to the Tukeys Test ( P <0.05). Error bars represent the standard error of the mean. Fi gure 18. Functional response of Zelus longipes adult females to increasing densities of Diaphorina citri adults.

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48 Figure 19. Preference of Zelus longipes 1st instar nymphs, males and females of Zelus longipes for Diaphorina citri and Tamarixia radiata adults. The preference index assigns preference values from 0 to 1, where 0.5 represents no preference. The position of in the bottom (0< <0.5) or top (0.5< values were considered significant when 95% confidence intervals (error bars) based on the t distribution did not overlap with =0.5 Zelus nymphs Zelus males Zelus femalesPreference index 0.0 0.2 0.4 0.6 0.8 1.0 Diaphorina citri Tamarixia radiata

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49 2D Graph 1 Zelus males Zelus femalesPreference index 0.0 0.2 0.4 0.6 0.8 1.0 Anastrepha suspensa Diaphorina citri Figure 110. Preference of males and females of Zelus longipes for Diaphorina citri and Anastrepha suspensa adults. The preference index assigns preference values from 0 to 1, where 0.5 represents no preference. The position of in the bottom (0< <0.5) or top (0.5< <1) of the graph shows preference for a particular prey. Mean values were conside red significant when 95% confidence intervals (error bars) based on the t distribution did not overlap with =0.5

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50 CHAPTER 3 ANTS ASSOCIATED WITH Diaphorina citri AND THEIR ROLE IN ITS BIOLOGICAL CONTROL Diaphorina citri Kuwayama is a serious threat to the citrus industry in Florida, not only because it extracts the sap of citrus plants and causes leaf distortion and sooty mold, but, most importantly, because it is associated with a bacterium that is the causal agent of probably the worst disease affecti ng citrus in the world, the citrus greening or huangblonbling (Halbert and Manjunath 2004, Tsai 2008). This insect invaded Florida in 1998 (Halbert et al. 2003 ). Citrus greening was reported in the state within 8 years of the arrival of D. citri (Bove 2006). A classical biological program was implemented with the importation, rearing and release of two specific parasitoids of D. citri the eulophid ectoparasitoid, Tamarixia radiata (Waterston), and the encyrtid endoparasitoid, Diaphorencyrtus aligharhensis ( Shafee, Alam and Agarwal .) (Hoy et al. 2001). Several surveys conducted after the release of the biological control agents showed that only T. radiata was established but caused low mortality of D. citri (Qureshi et al. 2009, Chong et al. 2010). These res ults were surprising when compared with the good performance of T. radiata in islands like Reunion, Guadaloupe, and Puerto Rico (Aubert et al. 1996, Etienne et al. 2001, Pluke et al. 2008). Several theories have been proposed for the lack of success of D citri parasitoids in Florida. Some of them relate the intensive use of insecticides affecting negatively T. radiata (Hall and Nguyen 2010), while others consider the genetic variability of the released parasitoid (Barr et al. 2009). Another reason to tak e into account, may be the interaction with other natural enemies present in the system. For instance, experimental data from central and south Florida support this point of view, suggesting that coccinellids are intraguild predators responsible for the poor performance of the parasitoid (Michaud 2004, Qureshi and Stansly 2009) T he

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51 results of the Chapter 2 of this thesis suggest that Zelus longipes ( L. ) might be an intraguild predator of T. radiata. An important, but poorly studied player in the complex of organisms involved in the D. citrigreening system are ants (Hymenoptera:Formicidae), which are well known for establishing mutualistic relationships with some hemipterans, either by tending their young or by using the hemipteran s (i.e., soft scales, ap hids) sugary excretions (i.e., honeydew) as a carbohydrate source. As a reward, the hemipterans receive ant protection against natural enemies (Way 1963, Way and Khoo 1992). This relationship can be obligatory when the ant and the honeydew producing hemipt eran have coevolved together and are interdependent with one another or facultative when the association is not indispensable for the survival of the hemipteran. The latter relationship is more common in nature than the obligatory mutualism (Delabie 2001). The mutualism between ants and hemipterans is frequent in aphids, mealybugs, and membracids and most of the published literature about this phenomenon addresses members of these families (Delabie and Fernandez 2003). The ant mutualism with psyllids have been less documented; one example is the relationship of the hawthorn psyllids Cacopsylla peregrina Forster, C. melanoneura Forster, C. crataegi (Schrank) with the ants Lasius niger (L.) and Formica pratensis Retzius that causes an increase in parasitism du e to the expelling of hyperparasitoids (Novak 1994). A second example is the mutualistic relationship of the ants Pheidole megacephala F.and Crematogaster striatula Emery, with the psyllid Diaphorina enderleini Klimaszewski in Africa. Workers of these ants build shelters in order to protect the immatures of the psyllid against natural enemies and environmental adversities (Alene et al. 2011). Several species of

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52 ants have been reported associated with D. citri in Florida. Michaud (2004) found Brachymyrmex ob scurior Forel, Camponotus floridanus (Buckley), Crematogaster ashmeadi (Mayr), Dorymyrmex reginicula (Trager), Monomorium floricola Jerdon, Paratrechina bourbonica (Forel), Pseudomyrmex gracilis (Fabricius), Solenopsis invicta Buren and Dorymyrmex bureni ( Trager) collecting honeydew from D. citri nymphs and also observed D. bureni carrying away D. citri nymphs on several occasions. Camponotus floridanus and Pheidole spp. were observed carrying nymphs in D. citri infested flushes of M. paniculata in south Fl orida (Pea et al. 2008). In the same area, Chong et al. (2010) found Crematogaster spp., Pheidole spp., Pseudomyrmex spp. and Solenopsis spp. associated with D. citri in M. paniculata. In this study I try to elucidate the role of ants in the biological co ntrol of D. citri by observing ant behavior and by determining if ant presence affects parasitism and psyllid densities in orange jasmine, M. paniculata and Persian lime, Citrus latifolia Materials and Methods All the experiments were conducted at the Uni versity of Florida, Tropical Research and Education Center, in Homestead Fl. (25.38N, 80.28W) Experiment 1. Identification and Behavior of Ants Related with D. citri in an Orange Jasmine Hedge Surveys were conducted on 11 Oct and 8 Nov 2011 with observati ons made every 2 h during a 24h period, beginning at 9 am and ending at 7 am on the next day. A 30 m long orange jasmine ( M paniculata) hedge was selected. Three 2.1m long plots distanced 2 m from each other were chosen. Five D. citri infested flushes w ere randomly chosen on each plot, and the ant number and behavior (i.e., tending nymphs, feeding on the nymphs excretions, walking and/or interacting with other natural enemies

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53 of ants) was recorded for a 2min period every two hr a headlamp was used duri ng the night observations Thereafter, each flush containing ants and D. citri nymphs was cut from the hedge, placed into in a 50m L vial, and brought to the laboratory for further examination under a microscope. The number of D. citri nymphs per flush was recorded; ants were sorted and sent to Dr. Mark Deyrup (Archbold Biological Station, Venus Fl) for identification. A one way analysis of variance (PROC GLM) was made comparing the number of ants present in the infested flushes per evaluation time. A reg ression analysis (PROC REG) was performed between the number of ants and the number of D. citri nymphs per flush (SAS Institute, Inc. 2001). Experiment 2. Ant Exclusion Experiments in M. paniculata and C latifolia This experiments were conducted in an 3 0 m long M. paniculata hedge and in twenty C. latifolia trees located at the Tropical Research and Education Center, Homestead, FL. Both hosts were hedged in order to promote flushing. M. paniculata was hedged on 20 Dec 2011 and C. latifolia was hedged on 10 March 2012. When leaf flushes reached 2 to 3 cm in length, 36 flushes infested with D. citri eggs were chosen, and one of the following treatments was randomly assigned to each shoot: a) Ant exclusion trough a 2 cm wide barrier of Tanglefoot (Tanglefoot Company Grand Rapids, MI) smeared at the base of each flush; b) Control: no Tanglefoot. The number of ants was counted, and the presence of predators and parasitoids of D. citri, was observed for 2min on each one of the selected flushes. The evaluations were performed every 2 d during a 30d period; ant specimens were sent to Dr. Mark Deyrup for their identification. Thirty days after treatment, when most of the D. citri nymphs infesting the flushes were in the fifth instar, all the 36 shoots were cut and brought to the laboratory where they were inspected under the microscope with the objective to count

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54 the number of parasitized and unparasitized nymphs and to calculate percent parasitism. Differences in percentages of parasitism by T. radiata in both tr eatments were compared using unpaired t test Test (PROCTTEST, SAS Institute, Inc. 2001). Experiment 3. Effect of Ant Control on D. citri Parasitism This experiment was conducted in a 4year old C. latifolia grove. Eighteen trees were selected and hedged in order to promote flushing on 16 May 2012. At the same time, one of the following treatments was applied to each tree: A) Extinguish Plus granular bait, Hydramethylnon 0.365%+ S Methoprene 0.250% (Wellmark International, Schaumburg, IL) (5 g/tree) applied to the soil surrounding the trunk, B) control, without the use of the granular bait. A buffer tree was left between the treated trees. Five flushes infested with D. citri eggs were selected and tagged in each tree; the number of ants and the species present in the flushes, as well as presence of other natural enemies, was observed for 2min per flush. The evaluations were repeated on 23 May, 27 May, 31 May, 4 Jun, 8 Jun, and 12 Jun 2012. On 12 Jun 2012, when most of the D. citri nymphs were in the fifth instar, the flushes were cut and brought to the laboratory in order to determine the percentage of parasitism by T. radiata The differences between population of ants and nymphs of D. citri and percentages of parasitism by T. radiata in both treatments were compared using unpaired t test (PROC TTEST SAS Institute, Inc. 2001). Results Experiment 1. Identification and Behavior of A nts R elated with D. citri in an Orange J asmine hedge Two ant species were identified during this survey, the bigheaded ant Pheidole megacephala Fabricius, and the rover ant Brachymyrmex obscurior Forel. Pheidole

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55 megacephala was the most common species during both surveys. Both species were active day and night. During the first survey the highest number of ants (n = 5) was observed at 7 pm; this number was significantly different from the evaluation at 9 am ( n= 1.4) ( F = 2.03; df = 11,168; P = 0.0288). No significant differences were observed ( F = 1.21; df = 11,168; P = 0.2843) during the second survey. The mean number of ants per fl ush varied from 3.33 (9 pm and 3 am) to 0.93 ants per flush at 1 pm (Fig. 21). Forty two percent of the 180 observed ants were seen feeding on the sugary excretion of the D. citri nymphs during the first evaluation; however, during the second evaluation o nly 39% were observed feeding. Ants were also observed walking (35% in the first evaluation and 44% in the second evaluation). No interactions with other arthropods or aggressive behavior against D. citri nymphs or adults were observed during the evaluations (Fig. 2 2). No statisticall correlation was found between the number of ants and the number of D. citri nymphs in the first ( b = 0.01, t (178) = 0.653, P = 0.5177) and second ( b = 0.02, t (178) = 0.97, P = 0.3876) evaluation (Fig. 2 3). Experiment 2. Ant E xclusion E xperiments in M. paniculata and C. latifolia Pheidole megacephala was the only ant species found in M. paniculata. P. megacephala, B. patagonicus and Solenopsis invicta were observed in C. latifolia The number of P. megacephala found in the unprotected flushes in M. paniculata fluctuated between 0.15 (6 Jan) and 0.5 (2 Jan and 8 Jan). In C. latifolia the number of pooled species of ants varied between 1.44 (31 Mar) and 6.61(25 Mar) (Fig. 24). During the first three evaluations, the most abundant species was P. megacephala but in the rest of the evaluations it was displaced by B. patagonicus (Fig. 2 5). In both hosts the flushes smeared with Tanglefoot remained free of ants during the experiment. No other arthropods were seen during the eval uations, except on 6 Jan when a Z. longipes

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56 female was observed feeding on the honeydew of a D. citri nymph in a Tanglefoot treated flush in M. paniculata; in C. latifolia a Z. longipes 5th instar nymph was seen in an unprotected flush in 2 Apr. In both experiments, only one worker of S. invicta was observed carrying a D. citri adult T his observation was made in an untagged flush in C. latifolia In untreated flushes, the number of D. citri nymphs was not significantly higher than in the flushes treated with Tanglefoot in M. paniculata ( t = 0.95; df = 34; P = 0.35) or C. latifolia ( t = 2.07; df = 34; P = 0.0513) (Fig. 2 6). However, i n both hosts the percentage of nymphs parasitized by T. radiata was significantly higher in the flushes where the ants were physically excluded by the use of the Tanglefoot sticky barrier. In M. paniculata treated flushes 20.36% of the nymphs were parasitized compared to 0.39% parasitism in the untreated control flushes ( t = 3.35; df = 34; P = 0.002). Fifthy eight percent of the psyllid nymphs were parasitized in the C. latifolia Tanglefoot treated flushes compared with 8.57% parasitism in the control ( t = 0.47; df = 34; P = 0.0003) (Fig. 27). Experiment 3. E ffect of A nt C ontrol on D. citri P arasitism The ant species P. megacephala and B. patagonicus were observed tending D. citri nymphs. No differences in ant densities were recorded until the last evaluation when the number of ants was significantly lower in the trees treated with the granular bait (1.22 ants per flush) compared with the untreated trees (4.02 ants per flush)( t = 2.3; df = 16; P = 0.036) (Fig. 28). In the treated trees, P. megacephala was the dominant species during the evaluations of 5 May, 4 Jun, 8 Jun and 12 Jun 2012 in the control trees this species was also the most common except during the evaluations of 31 May and 6 Jun 2012 in which B. patagonicus was the most abundant species (Fig. 29). The number of D. citri nymphs was not statistically higher in the control trees compared with

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57 the trees treate d with Extinguish P lus ( t = 1.40; df = 16; P = 0.1792) (Fig. 210) The percentage of parasitism by T. radiata was statistically higher (19.56%) in the trees treated with the granular bait in comparison with the control trees which had in average 3.62% parasitism ( t = 1.40 ; df = 16; P = 0.1792) (Fig. 2 11). Discussion In this study we found four species of ants tending D. citri; Pheidole megacephala and Brachymyrmex obscurior in M. paniculata, and P. megacephala, B. patagonicus and S. invicta in C. latifolia Pheidole megacephala and Brachymyrmex obscurior have been previously reported tending D. citri (Michaud 2004, Pea et al. 2008). Here I report for the first time Brachymyrmex patagonicus tending D. citri. All of the ant species are invasive species in USA. Pheidole megacephala is native of Africa and the other two species are natives of South America. Pheidole megacephala is an omnivorous opportunistic species with a broad range of food preferences such as tending sapsucking Hemiptera and eating their sugary excretions (Deyrup et al. 200 0 MacGown et al 2007) This behavior was confirmed by our observations in both hosts. The species involved in this system have been found tending other hemipterans, i.e. B. osbcurior tending Dalbulus quinquenotatus Del ong and Nault (Cicadellidae) (Larsen et al. 1991), B. patagonicus tending Aphis gossypii Glover (Aphididae) (Barnum 2008), Pheidole megacephala tending Coccus viridis (Coccidae) (Reimer et al. 1993), and S. invicta tending Phenacoccus solenopsis Tinsley (P seudococcidae) (Zhou et al. 2012). We observed that P. megacephala and B. obscurior can forage in the infested flushes during night and day. This report is in agreement with the observations made by Cogni and Freitas (2002), Dejean et al. (2007) and Bestel meyer (2008).

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58 The ant exclusion experiments in both host plants showed that the ant presence in the flushes infested by D. citri has a negative effect on the performance of the parasitoid T. radiat a Although we do not registered a direct interaction between ants and the parasitoid, the difference in percentage of parasitism was significant. On the other hand we did not see any aggressive behavior of ants against nymphs of D. citri, and the number of D. citri nymphs in both treatments did not show a statist ical difference, therefore, we assume that in this experiment the ants did not eat the tended psyllids. According to Way (1963), ants tending Hemiptera can feed on their protected trophobionts if the source of proteins and lipids is scarce. Qureshi and Sta nsly (2009) also found a higher percentage of parasitism by T. radiata in flushes protected with a sticky barrier in comparison with other exclusion treatments in Citrus sinensis L but they did not associate the ants with this phenomenon. In the last experiment, we proved that the use of granular bait can help to reduce ant populations and consequently increase the percentage of parasitism. In this trial we could not totally avoid the access of ants to D. citri nymphs like we did in the previous experiment using Tanglefoot, however, we could decrease ant populations during the most susceptible stage for parasitism of D. citri (4th to 5th instar), and apparently this was enough to increase the activity of T. radiata. For instance, the use of different kinds of chemical treatments has been tested with success in the control of ants interfering with parasitoids of the grape mealybug in California and South Africa (Addison 2002, Klotz et al. 2003). We encourage similar studies in Florida citrus to determine if ant control in this agroecosystem will be a practical IPM practice against D. citri.

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59 Figure 21. Number of ants found in Murraya paniculata flushes infested with Diaphorina citri during a 24h period Pheidole megacephala (99% of the observations) and B rachymyrmex obscurior (1% of the observations) Oct 11 (A) and Nov 11 (B). Different letters represent a significant difference (Tukeys Test, P < 0.05). Error bars represent the standard error of the mean.

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60 Figure 22 Behavior of ants registered in D iaphorina citri infested flushes Pheidole megacephala (99% of the observations) and Brachymyrmex obscurior (1% of the observations) Oct 11 (A) and Nov11 (B).

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61 Figure 23. Relationship between number of ants and number of nymphs of Dia phorina citri in Murraya paniculata. Oct11 (A) and Nov11 (B).

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62 Figure 24. Number of ants found in unprotected flushes Murraya paniculata ( Pheidole megacephala 100 % of the observations) (A) and Citrus latifolia ( Brachymyrmex patagonicus 55%, P. megacephala 29% and Solenopsis invicta 15 % of the observations) (B). Error bars represent the standard error of the mean.

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63 Figure 25. Proportion of ants species found in unprotected flushes in Citrus latifolia

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64 Figure 26. Number of Diaphorina citri n ymphs present in flushes with and without ant exclusion treatment Murraya paniculata (A) and Citrus latifolia (B). Error bars represent the standard error of the mean.

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65 Figure 27. Percentage parasitism by Tamarixia radiata in Diaphorina citri infes ted flushes with and without ant exclusion. Murraya paniculata (A) and Citrus latifolia (B). Error bars represent the standard error. Bars marked with indicate significant differences (independent t test, P <0.05)

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66 Figure 28. Number of ants found in flushes of trees with and without the use of granular insecticidal ant bait in Citrus latifolia Error bars represent the standard error of the mean. Different letters represent significant differences, ns means no significant differences (independent t test, P <0.05).

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67 Figure 29. Proportion of ants species found in flushes of trees with and without t he use of granular ant bait in Citrus latifolia Extinguish Plus (A) Control (B).

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68 Fig ure 2 10. Number of Diaphorina citri nymphs present in flushes of trees with and without the use of granular ant bait in Citrus latifolia Error bars represent the standard error. Figure 211. Percentage parasitism by Tamarixia radiata in Diaphorina citri infested flushes of trees with and without the use of granular ant bait in Citrus latifolia Error bars represent the standard error. Bars marked with indicate significant differences (independent t test, P <0.05)

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69 CHAPTER 4 CONCLUSIONS The objective of this thesis was to study interactions between some of the natural enemies of D. citri in south Florida. The first study evaluated the effect of different densities of Zelus longipes on the population of D. citri and the parasitoid T. radiata under controlled conditions The study showed that Z. longipes has potential for controlling adults of D. citri. N ymphs and adults of Z. longipes were efficient preying on adults of D. citri but the effect on psyllid nymphs was less notorious. The adult females showed a Type III functional response to psy llid adults. T he predator was also efficient consuming adults of T. radiata. The prey preferences studies of the behavior of 1st intar nymphs of Z. longipes showed a preference for the parasitoid over the psyllid. With these antecedents, field experiments are needed to find if intraguild predation occurs outdoors and how this relates to reduce the effectiveness of parasitoids. The second study dealt with the role of ants in the biological control of D. citri. An assemblage of four species was found associated with D. citri in orange jasmine and Persian lime. The behavior study showed that these ants are active day and night and that they use the psyllid wax as a food source. The ant exclusion experiments suggest that ants are affecting the performance of T. radiata and that the use of chemical bait can help to improve the percentage of parasitism. More estudies are needed to determine economic ways to exclude ants in Florida citrus orchards.

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82 BIOGRA PH ICAL SKETCH Bernardo Navarrete was born and raised in Portoviejo, Ecuador. He got his bachelors degree in agronomic engineering in the Universidad Tcnica de Manab in 1999. Since his graduation, Bernardo has been working as an assistant researcher in the entomology departm ent of the Portoviejo Experimental Station which belongs to the National Institute of Agriculture Research of Ecuador (INIAP). During his more than 10 years working in research, Bernardo has collaborated in IPM projects on vegetables, corn and citrus. In 2010 he received a scholarship from INIAP for pursuing a Master of Science degree in the University of Florida. In his spare time Bernardo enjoys taking photographies of insects in their natural environment.